Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody

ABSTRACT

New combined therapeutic regimens for treatment of B-cell lymphomas are disclosed which comprise in particular administration of anti-CD20 antibodies to patients having low-, intermediate- or high-grade non-Hodgkins lymphomas.

CROSS-REFERENCE TO RELATED APPLICATION

This application take priority from Provisional Application No.60/096,180, filed on Aug. 11, 1998; the entire content of which ishereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the use of anti-CD20 antibodies or fragmentsthereof in the treatment of B-cell lymphomas, particularly the use ofsuch antibodies and fragments in combined therapeutic regimens.

BACKGROUND OF THE INVENTION

The use of antibodies to the CD20 antigen as diagnostic and/ortherapeutic agents for B-cell lymphoma has previously been reported.CD20 is a useful marker or target for B-cell lymphomas as this antigenis expressed at very high densities on the surface of malignant B-cells,i.e., B-cells wherein unabated proliferation can lead to B-celllymphomas.

CD20 or Bp35 is a B-lymphocyte-restricted differentiation antigen thatis expressed during early pre-B-cell development and remains untilplasma cell differentiation. It is believed by some that the CD20molecule may regulate a step in the B-cell activation process which isrequired for cell cycle initiation and differentiation. Moreover, asnoted, CD20 is usually expressed at very high levels on neoplastic(“tumor”) B-cells. The CD20 antigen is appealing for targeted therapy,because it does not shed, modulate, or internalize.

Previous reported therapies involving anti-CD20 antibodies have involvedthe administration of a therapeutic anti-CD20 antibody either alone orin conjunction with a second radiolabeled anti-CD20 antibody, or achemotherapeutic agent.

In fact, the Food and Drug Administration has approved the therapeuticuse of one such anti-CD20 antibody, Rituxan®, for use in relapsed andpreviously treated low-grade non-Hodgkin's lymphoma (NHL). Also, the useof Rituxan® in combination with a radiolabeled murine anti-CD20 antibodyhas been suggested for the treatment of B-cell lymphoma.

However, while anti-CD20 antibodies and, in particular, Rituxan® (U.S.;in Britain, MabThera®; in general Rituximab®), have been reported to beeffective for treatment of B-cell lymphomas, such as non-Hodgkin'slymphoma, the treated patients are often subject to disease relapse.Therefore, it would be beneficial if more effective treatment regimenscould be developed. More specifically, it would be advantageous ifanti-CD20 antibodies had a beneficial effect in combination with otherlymphoma treatments, and if new combined therapeutic regimens could bedeveloped to lessen the likelihood or frequency of relapse. Also, itwould be helpful if current treatment protocols for B-cell lymphoma wereimproved whereby patients with lymphomas which are refractory to othertreatment methods could be treated with chimeric or radiolabeledanti-CD20 antibodies. It would also be helpful if treatment withanti-CD20 antibodies, particularly in combination with other treatments,could be used as therapy for other types of lymphoma besides low grade,follicular non-Hodgkins lymphoma (NHL).

SUMMARY OF THE INVENTION

The present invention discloses combined therapeutic treatments forB-cell lymphomas, and reports the benefits of treating relapsed orrefractory B-cell lymphomas with chimeric and radiolabeled anti-CD20antibodies. In particular, it has been found that treatment withanti-CD20 antibody provides a beneficial synergistic effect whenadministered in combination with cytokines, radiotherapy, myeloablativetherapy, or chemotherapy. Surprisingly, patients who had prior bonemarrow or stem cell transplantation had an unexpected increase in theover-all response rate when compared with patients with no priortherapy.

DETAILED DESCRIPTION OF THE INVENTION

This invention encompasses combined therapeutic regimens for thetreatment of B-cell lymphomas. In general, such methods include a methodfor treating relapsed B-cell lymphoma, where a patient having priortreatment for lymphoma has relapsed and is administered atherapeutically effective amount of a chimeric anti-CD20 antibody. Suchprior treatments can include, for example, previous treatment withanti-CD20 antibodies, treatments which included a bone marrow or stemcell transplantation, radiotherapy and chemotherapy. The previouschemotherapy may be selected from a wide group of chemotherapeuticagents and combination regimens, including CHOP, ICE, Mitozantrone,Cytarabine, DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La Lachemotherapy regime, ABVD, CEOP, 2-CdA, FLAG & IDA with or withoutsubsequent G-CSF treatment), VAD, M & P, C-Weekly, ABCM, MOPP and DHAP.

Also included in the methods of the invention are methods for treating asubject having B-cell lymphoma wherein the subject is refractory forother therapeutic treatments, including all those listed above, i.e.,treatment with chimeric anti-CD20 antibody, treatments which included abone marrow or stem cell transplantation, radiotherapy and chemotherapy.In particular, encompassed are methods of treating a patient who has notexhibited appreciable tumor remission or regression after administrationof a chimeric anti-CD20 antibody, comprising administering to saidpatient a radiolabeled anti-CD20 antibody.

In particular, the methods of treating a patient with a radiolabeledantibody after a chimeric antibody are performed whereby theradiolabeled anti-CD20 antibody is administered from about one week toabout two years after said administration of said chimeric anti-CD20antibody. More particularly, the radiolabeled anti-CD20 antibody isadministered from about one week to about nine months after saidadministration of said chimeric anti-CD20 antibody.

While any anti-CD20 antibodies can be used for the methods of thepresent invention, a preferred chimeric antibody is C2B8 (IDECPharmaceuticals, Rituximab®). A preferred radiolabeled antibody is Y2B8,which is a murine antibody labeled with yttrium-90 (⁹⁰Y). However,antibodies with other radiolabels may be used, particularly thoselabeled with a beta or alpha isotope. Anti-CD19 antibodies may also beused.

One of skill in the art would know the parameters for choosing aparticular type of anti-CD20 antibody. For instance, chimeric andhumanized antibodies are beneficial for decreased immunogenicity, andfor facilitating antibody effector mediated immune reactions via thehuman constant region domains. Murine and other mammalian antibodies, incontrast, are beneficial for delivering a radiolabel to the tumor cell,as such antibodies generally have a decreased half-life in vivo.

Antibody treatments performed initially to which patients are refractoryor have relapsed may include initial treatments with chimeric antibodiesor mammalian antibodies. Also encompassed are initial treatments withother antibodies, including anti-CD19 antibodies and anti-Lymantibodies, and treatments with antibodies labeled with cytotoxicmoieties, such as toxins, and radiolabels, e.g., Oncolym® (Techniclone)or Bexxar (Coulter).

It should be clear that the combined therapeutic regimens of the presentinvention can be performed whereby said therapies are givensimultaneously, i.e., the anti-CD20 antibody is administeredconcurrently or within the same time frame (i.e., the therapies aregoing on concurrently, but the agents are not administered precisely atthe same time). The anti-CD20 antibodies of the present invention mayalso be administered prior to or subsequent to the other therapies.Sequential administration may be performed regardless of whether thepatient responds to the first therapy to decrease the possibility ofremission or relapse.

The combined therapies of the present invention include a method fortreating B-cell lymphoma comprising administering at least one chimericanti-CD20 antibody and at least one cytokine. In particular, theinvention includes a method for treating B-cell lymphoma comprisingadministering a synergistic therapeutic combination comprising at leastone anti-CD20 antibody and at least one cytokine, wherein thetherapeutic effect is better than the additive effects of either therapyadministered alone. Preferred cytokines are selected from the groupconsisting of alpha interferon, gamma interferon, IL-2, GM-CSF andG-CSF. Again, the anti-CD20 antibody and the cytokine(s) may beadministered sequentially, in either order, or in combination.

Also included in the present invention is a method for treating B-celllymphoma comprising administering to a patient a therapeuticallyeffective amount of a chimeric anti-CD20 antibody before, during orsubsequent to a chemotherapeutic regimen. Such a chemotherapy regimenmay be selected from the group consisting of, at the very least, CHOP,ICE, Mitozantrone, Cytarabine, DVP, ATRA, Idarubicin, hoelzerchemotherapy regime, La La chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG& IDA with or without subsequent G-CSF treatment), VAD, M & P, C-Weekly,ABCM, MOPP and DHAP.

Also encompassed are methods for treating B-cell lymphoma comprisingadministering to a patient a therapeutically effective amount of achimeric anti-CD20 antibody before, during or subsequent to a bonemarrow or peripheral stem cell transplant. Such bone marrow transplantmay also be accompanied by other therapeutic regimens such aschemotherapy. The antibodies of the present invention may also be usedin a method of reducing residual CD20+ tumor cells in bone marrow orstem cells before or after myeloablative therapy by administering to apatient a chimeric anti-CD20 antibody. It may also be possible to usesuch antibodies in vitro to induce apoptosis of tumor cells and reduceor cure bone marrow or stem cell preparations of residual tumor cellsbefore they are infused back into the patient.

It should be understood that stem cell transplants may be allogeneic orautologous. If the transplant is allogeneic, i.e., from another person,the disclosed therapeutic regimens may include treatments withimmunosuppressive drugs before administration of the anti-CD20antibodies. Coadministration of other drugs designed to enhanceacceptance of the transplant and stimulate the production anddifferentiation of immune cells is also contemplated. For instance, ithas been shown that administration of GM-CSF to marrow transplantrecipients promotes the development of specific bone marrow cells whichin turn produces circulating infection-fighting neutrophils, andincreased the survival rate of marrow transplant recipients.

The methods of the present invention may be used to treat a variety ofB-cell lymphomas, including low grade/follicular non-Hodgkin's lymphoma(NHL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL,intermediate grade diffuse NHL, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL and Waldenstrom's Macroglobulinemia. It should be clear to those ofskill in the art that these lymphomas will often have different namesdue to changing systems of classification, and that patients havinglymphomas classified under different names may also benefit from thecombined therapeutic regimens of the present invention.

For instance, a recent classification system proposed by European andAmerican pathologists is called the Revised European American Lymphoma(REAL) Classification. This classification system recognizes Mantle celllymphoma and Marginal cell lymphoma among other peripheral B-cellneoplasms, and separates some classifications into grades based oncytology, i.e., small cell, mixed small and large, large cell. It willbe understood that all such classified lymphomas may benefit from thecombined therapies of the present invention.

The U.S. National Cancer Institute (NCI) has in turn divided some of theREAL classes into more clinically useful “indolent” or “aggressive”lymphoma designations. Indolent lymphomas include follicular celllymphomas, separated into cytology “grades,” diffuse small lymphocyticlymphoma/chronic lymphocytic leukemia (CLL),lymphoplasmacytoid/Waldenstrom's Macroglobulinemia, Marginal zonelymphoma and Hairy cell leukemia. Aggressive lymphomas include diffusemixed and large cell lymphoma, Burkitt's lymphoma/diffuse smallnon-cleaved cell lymphoma, Lymphoblastic lymphoma, Mantle cell lymphomaand AIDS-related lymphoma. These lymphomas may also benefit from thecombined therapeutic regimens of the present invention.

Non-Hodgkin's lymphoma has also been classified on the basis of “grade”based on other disease characteristics including low-grade,intermediate-grade and high-grade lymphomas. Low-grade lymphoma usuallypresents as a nodal disease, and is often indolent or slow-growing.Intermediate- and high-grade disease usually presents as a much moreaggressive disease with large extranodal bulky tumors. Intermediate- andhigh-grade disease, as well as low grade NHL, may benefit from thecombined therapeutic regimens of the present invention.

The Ann Arbor classification system is also commonly used for patientswith NHL. In this system, stages I, II, III, and IV of adult NHL can beclassified into A and B categories depending on whether the patient haswell-defined generalized symptoms (B) or not (A). The B designation isgiven to patients with the following symptoms: unexplained loss of morethan 10% body weight in the 6 months prior to diagnosis, unexplainedfever with temperatures above 38° C. and drenching night sweats.Occasionally, specialized staging systems are used:

Stage I—involvement of a single lymph node region or localizedinvolvement of a single extralymphatic organ or site.

Stage II—involvement of two or more lymph node regions on the same sideof the diaphragm or localized involvement of a single associatedextralymphatic organ or site and its regional lymph nodes with orwithout other lymph node regions on the same side of the diaphragm.

Stage III—involvement of lymph node regions on both sides of thediaphragm, possibly accompanying localized involvement of anextralymphatic organ or site, involvement of the spleen, or both.

Stage IV—disseminated (multifocal) involvement of 1 or moreextralymphatic sites with or without associated lymph node involvementor isolated extralymphatic organ involvement with distant (non-regional)nodal involvement.

For further details, see The International Non-Hodgkin's LymphomaPrognostic Factors Project: A predictive model for aggressivenon-Hodgkin's lymphoma. New England J. Med. 329(14): 987-994 (1993).

Preferred antibodies, dosage regimens and particular combinations oftherapy will now be illustrated by way of the following exemplary data.

Rituximab® and Y2B8

Non-Hodgkin's lymphoma (NHL) affects approximately 250,000 people in theUnited States. The majority of patients with NHL are not cured bychemotherapy, radiotherapy, or high-dose treatment with autologous bonemarrow (ABMT) or peripheral blood stem cell (PBSC) support.

Approximately 80% of non-Hodgkin's lymphomas are B-cell malignanciesand >95% of these express the CD20 antigen on the cell surface. Thisantigen is an attractive target for immunotherapy because it is foundexclusively on B-cells, and not on hematopoietic stem cells,pro-B-cells, normal plasma cells, or other normal tissues. It is notshed from the cell surface and does not modulate upon antibody binding(1).

Rituximab® is one of a new generation of monoclonal antibodies developedto overcome limitations encountered with murine antibodies, includingshort half-life, limited ability to stimulate human effector functions,and immunogenicity (2,3).

Rituximab® is a genetically engineered monoclonal antibody with murinelight- and heavy-chain variable regions and human gamma I heavy-chainand kappa light-chain constant regions. The chimeric antibody iscomposed of two heavy chains of 451 amino acids and two light chains of213 amino acids and has an approximate molecular weight of 145 kD.Rituximab® is more effective than its murine parent in fixing complementand mediating ADCC, and it mediates CDC in the presence of humancomplement (4). The antibody inhibits cell growth in the B-cell linesFL-18, Ramos, and Raji, sensitizes chemoresistant human lymphoma celllines to diphtheria toxin, ricin, CDDP, doxorubicin, and etoposide, andinduces apoptosis in the DHL-4 human B-cell lymphoma line in adose-dependent manner (5). In humans, the half-life of the antibody isapproximately 60 hours after the first infusion and increases with eachdose to 174 hours after the fourth infusion. The immunogenicity of theantibody is low; of 355 patients in seven clinical studies, only three(<1%) had a detectable anti-chimeric antibody (HACA) response.

Rituximab® was genetically engineered using the murine 2B8 antibody. The2B8 antibody has also been conjugated to different radiolabels fordiagnostic and therapeutic purposes. To this end, copending applicationSer. Nos. 08/475,813, 08/475,815 and 08/478,967, herein incorporated byreference in their entirety, disclose radiolabeled anti-CD20 conjugatesfor diagnostic “imaging” of B-cell lymphoma tumors before administrationof therapeutic antibody. “In2B8” conjugate comprises a murine monoclonalantibody, 2B8, specific to human CD20 antigen, that is attached toIndium[1,1,1] (¹¹¹In) via a bifunctional chelator, i.e., MX-DTPA(diethylene-triaminepentaacetic acid), which comprises a 1:1 mixture of1-isothiocyanatobenzyl-3-methyl-DTPA and1-methyl-3-isothiocyanatobenzyl-DTPA. Indium-[111] is selected as adiagnostic radionuclide because it emits gamma radiation and finds priorusage as an imaging agent.

Patents relating to chelators and chelator conjugates are known in theart. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed topolysubstituted diethylenetriaminepentaacetic acid chelates and proteinconjugates containing the same, and methods for their preparation. U.S.Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow alsorelate to polysubstituted DTPA chelates. These patents are incorporatedherein in their entirety.

The specific bifunctional chelator used to facilitate chelation inapplication Ser. Nos. 08/475,813, 08/475,815 and 08/478,967 was selectedas it possesses high affinity for trivalent metals, and provides forincreased tumor-to-non-tumor ratios, decreased bone uptake, and greaterin vivo retention of radionuclide at target sites, i.e., B-cell lymphomatumor sites. However, other bifunctional chelators are known in the artand may also be beneficial in tumor therapy.

Also disclosed in application Ser. Nos. 08/475,813, 08/475,815 and08/478,967 are radiolabeled therapeutic antibodies for the targeting anddestruction of B-cell lymphomas and tumor cells. In particular, the Y2B8conjugate comprises the same anti-human CD20 murine monoclonal antibody,2B8, attached to yttrium-[90] (⁹⁰Y) via the same bifunctional chelator.This radionuclide was selected for therapy for several reasons. The 64hour half-life of ⁹⁰Y is long enough to allow antibody accumulation bythe tumor and, unlike e.g. ¹³¹I, it is a pure beta emitter of highenergy with no accompanying gamma irradiation in its decay, with a rangeof 100 to 1000 cell diameters. The minimal amount of penetratingradiation allows for outpatient administration of ⁹⁰Y-labeledantibodies. Furthermore, internalization of labeled antibodies is notrequired for cell killing, and the local emission of ionizing radiationshould be lethal for adjacent tumor cells lacking the target antigen.

Because the ⁹⁰Y radionuclide was attached to the 2B8 antibody using thesame bifunctional chelator molecule MX-DTPA, the Y2BS conjugatepossesses the same advantages discussed above, e.g., increased retentionof radionuclide at a target site (tumor). However, unlike ¹¹¹In, itcannot be used for imaging purposes due to the lack of gamma radiationassociated therewith. Thus, a diagnostic “imaging” radionuclide, such as¹¹¹In, can be used for determining the location and relative size of atumor prior to and/or following administration of therapeutic chimericor ⁹⁰Y-labeled antibodies in the combined regimens of the invention.Additionally, indium-labeled antibody enables dosimetric assessment tobe made.

Depending on the intended use of the antibody, i.e., as a diagnostic ortherapeutic reagent, other radiolabels are known in the art and havebeen used for similar purposes. For instance, radionuclides which havebeen used in clinical diagnosis include ¹³¹I, ¹²⁵I, ¹²³I, ⁹⁹Tc, ⁶⁷Ga, aswell as ¹¹¹In. Antibodies have also been labeled with a variety ofradionuclides for potential use in targeted immunotherapy (Peirersz etal. (1987) The use of monoclonal antibody conjugates for the diagnosisand treatment of cancer. Immunol Cell Biol. 65: 111-125). Theseradionuclides include ¹⁸⁸Re and ¹⁸⁶Re as well as ⁹⁰Y, and to a lesserextent ¹⁹⁹Au and ⁶⁷Cu. I-(131) has also been used for therapeuticpurposes. U.S. Pat. No. 5,460,785 provides a listing of suchradioisotopes and is herein incorporated by reference.

As reported in copending application Ser. Nos. 08/475,813, 08/475,815and 08/478,967, administration of the radiolabeled Y2B8 conjugate, aswell as unlabeled chimeric anti-CD20 antibody, resulted in significanttumor reduction in mice harboring a B-cell lymphoblastic tumor.Moreover, human clinical trials reported therein showed significantB-cell depletion in lymphoma patients infused with chimeric anti-CD20antibody. In fact, chimeric 2B8 has recently been heralded the nation'sfirst FDA-approved anti-cancer monoclonal antibody under the name ofRituxan®. Thus, at least one chimeric anti-CD20 antibody has been shownto demonstrate therapeutic efficacy in the treatment of B-cell lymphoma.

In addition, U.S. application Ser. No. 08/475,813, herein incorporatedby reference, discloses sequential administration of Rituxan®, achimeric anti-CD20, with both or either indium-labeled oryttrium-labeled murine monoclonal antibody. Although the radiolabeledantibodies used in these combined therapies are murine antibodies,initial treatment with chimeric anti-CD20 sufficiently depletes theB-cell population such that the HAMA response is decreased, therebyfacilitating a combined therapeutic and diagnostic regimen.

Thus, in this context of combined immunotherapy, murine antibodies mayfind particular utility as diagnostic reagents. Moreover, it was shownin U.S. application Ser. No. 08/475,813 that a therapeutically effectivedosage of the yttrium-labeled anti-CD20 antibody followingadministration of Rituxan® is sufficient to (a) clear any remainingperipheral blood B-cells not cleared by the chimeric anti-CD20 antibody;(b) begin B-cell depletion from lymph nodes; or (c) begin B-celldepletion from other tissues.

Thus, conjugation of radiolabels to cancer therapeutic antibodiesprovides a valuable clinical tool which may be used to assess thepotential therapeutic efficacy of such antibodies, create diagnosticreagents to monitor the progress of treatment, and devise additionaltherapeutic reagents which may be used to enhance the initialtumor-killing potential of the chimeric antibody. Given the provenefficacy of an anti-CD20 antibody in the treatment of non-Hodgkin'slymphoma, and the known sensitivity of lymphocytes to radioactivity, itwould be highly advantageous for such chimeric and radiolabeledtherapeutic antibodies to find use in combined therapeutic regimenswhich decrease the frequency of relapsed or refractory non-Hodgkin'slymphoma. In addition, it would be beneficial if such combinedtherapeutic regimens found use in the treatment of other B-celllymphomas.

Low-Grade or Follicular NHL

Single-Agent Studies with Relapsed or Refractory NHL

FDA approval of Rituximab® was based on five single-agent studiesprimarily in patients with low-grade or follicular NHL. An early Phase Istudy of single Rituximab® infusions ranging from 10-500 mg/m²demonstrated that the maximum tolerated dose had not been reached;however, the length of infusion time at the highest dose was notconsidered feasible for outpatient therapy. The ORR in 15 patients was13% (Table 1)(6). TABLE 1 Rituximab ®: Summary of Efficacy ResultsMedian Median DR TIP Study Description Indication N* ORR CR PR (months)(months) References Phase I/II, Single-Dose Relapsed B-Cell Lymphoma 15 2 (13%) 0 (0%)  2 (13%) NA† 8.1  6 Single Agent Phase I/II,Multiple-Dose Relapsed Low-, Intermediate-, 34 17 (50%) 3 (9%) 14 (41%)8.6 10.2   7 Dose-Ranging and High-Grade Lymphoma Phase II;Multiple-Dose Newly Diagnosed and Relapsed 38  38 (100%) 22 (58%) 16(42%) 35.3+ 36.7+ 21, 22 Combined with CHOP Low-Grade or FollicularB-Cell Lymphoma Phase III, Multiple-Dose Relapsed Low-Grade or 151 76(50%) 9 (6%) 67 (44%) 11.6  13.2  8, 9 Single-Agent Follicular B-CellLymphoma Phase II, Multiple-Dose Relapsed Low-Grade or 35 21 (60%)  5(14%) 16 (46%) 13.4+ 19.4+ 13 Single-Agent Follicular B-Cell LymphomaPhase II, Multiple-Dose, Relapsed Low-Grade or 38 17 (45%)  4 (11%) 13(34%) 22.3+ 25.2+ 29 Combined with Interferon Follicular B-Cell LymphomaPhase II, Multiple-Dose, Relapsed Low-Grade or 28 12 (43%) 1 (4%) 11(39%) 5.9 8.1 14 Single-Agent Follicular B-Cell Lymphoma, Bulky DiseasePhase II, Multiple-Dose, Relapsed Low-Grade or 57 23 (40%)  6 (11%) 17(29%) 15.0+ 16.7+ 19, 20 Single-Agent Follicular B-Cell Lymphoma,Retreatment Phase II, Multiple-Dose Previously Untreated 30 29 (96%) 19(63%) 10 (33%) 11+  17+  34 Combined with CHOP Intermediate- orHigh-Grade Modality Lymphoma Phase II, Alternative Intermediate- orHigh-Grade 54 17 (32%) 5 (9%) 12 (22%) NA†  8.2+ 33 Multiple DosingB-Cell Lymphoma*N = number of evaluable patients†Not available

In Phase I of a Phase I/II dose-ranging study, patients received 125-375mg/m² administered as four weekly infusions. No dose-related toxicitieswere demonstrated, and 375 mg/m² was chosen as the Phase II dose. Tumorregressions were observed in 17 of 37 (46%) patients who received hisdose, including 3 (8%) complete responses (CR) and 14 (38%) partialresponses PR (7).

A subsequent single-arm pivotal study of Rituximab® infused at 375 mg/m²weekly times four was conducted in 166 patients with relapsed orrefractory, low-grade or follicular NHL (International WorkingFormulation [IWF] Types A-D and REAL classification, small lymphocyticlymphoma, follicular center, follicular Grades I, II, III (8)). Patientswith tumor masses >10 cm or with >5000 lymphocytes/μL in the peripheralblood were excluded from this study. The median age was 58 years (105men and 61 women) and the median number of prior treatments was three.Bone marrow involvement was present in 56% of 149 patients evaluated.Forty-five percent had ≧2 extranodal sites and 41% had bulky disease (≧5cm).

Complete response required the regression of all lymph nodes to <1×1 cm²demonstrated on two occasions at least 28 days apart on neck, chestabdomen, and pelvic CT scans, resolution of all symptoms and signs oflymphoma, and normalization of bone marrow, liver, and spleen. Partialresponse required a ≧50% decrease in the sum of the products ofperpendicular measurements of lesions without any evidence ofprogressive disease for at least 28 days. Patients who did not achieve aCR or PR were considered non-responders, even if a net decrease (>50%)of measurable disease was observed. Time to progression was measuredfrom the first infusion until progression.

The overall response rate (ORR) was 48% with a 6% CR and a 42% PR rate(8). The median time to progression (TTP) for responders was 13.2 monthsand the median duration of response (DR) was 11.6 months. Twenty-two of80 (28%) responders remain in ongoing remission at 20.9+ to 32.9+ months(9).

Administration of Rituximab® resulted in a rapid and sustained depletionof B-cells. Circulating B-cells were depleted within the first threedoses with sustained depletion for up to six to nine monthspost-treatment in 83% of patients. Median B-cell levels returned tonormal by 12 months following treatment. Although median NK cell countsremained unchanged, a positive correlation was observed between higherabsolute NK cell counts at baseline and response to Rituximab® (10).

Several baseline prognostic factors were analyzed to determine theircorrelation to response. Significantly, in 23 patients relapsed afterABMT or PBSC, the ORR was 78% versus 43% in patients who did not undergoprior high-dose therapy (p<0.01). In a multivariate analysis, the ORRwas higher in patients with follicular NHL as compared with smalllymphocytic lymphoma (58% vs. 12%, p<0.01), and higher in patients withchemosensitive relapse as compared with chemoresistant relapse (53% vs.36%, p=0.06). No effect on response rate was associated with: age >60years, extranodal disease, prior anthracycline therapy, or bone marrowinvolvement.

A statistically significant correlation was found between the medianserum antibody concentration and response at multiple time points duringtreatment and follow up (11).

Serum levels of antibody were higher in patients with follicular NHLcompared with small lymphocytic lymphoma. Mean serum antibody was alsoinversely correlated with measurements of tumor bulk and with the numberof circulating B-cells at baseline. The association of lower serumantibody concentrations with higher numbers of circulating NHL cells andwith higher tumor bulk suggest that the main mode of antibody clearanceis to tumor cells. The association of high serum antibody concentrationswith response and lower tumor bulk or circulating cells suggests thathigher or more doses of Rituximab® may be necessary to induce responsesin some subsets of patients, such as those with bulky disease.

Nevertheless, responses were seen with Rituximab® in 43% of patientswith tumors >5 cm and in 35% of patients with tumors >7 cm, suggestingthat treatment of patients with bulky disease with Rituximab® isfeasible. This is surprising considering it was long thought thatantibody therapy is not conducive to treating bulky disease due to thecompact nature of the tumors.

In a study conducted in Japan (12), patients with relapsed B-celllymphoma were treated with either 250 mg/m² (N=4) or 375 mg/m² (N=8) ofRituximab® weekly times four. Of 11 evaluable patients, 8 had follicularNHL, 2 had diffuse large-cell NHL, and one had mantle-cell lymphoma. Twoof the 11 had a CR and 5 had a PR for an ORR of 64%; all responders hadfollicular histology.

Because Rituximab® serum levels and response were positively correlatedin previous studies, a Phase II study of eight weekly doses of 375 mg/m²Rituximab® was conducted in low-grade or follicular NHL patients. TheORR was 60% in evaluable patients, with a 14% CR and a 46% PR rate.Median values for TTP in responders and DR were 13.4+ months and 19.4+months, respectively (13). Though it is difficult to compare acrossstudies, it appears that TTP and DR may be improved by using more doses.

Contrary to early assumptions about antibody therapy being useful onlyin micrometastatic disease, Rituximab® is quite active in high bulkdisease. In a separate study, 31 patients with relapsed or refractory,bulky low-grade NHL (single lesion of >10 cm in diameter) received 375mg/m² Rituximab® as four weekly infusions. Twelve of 28 evaluablepatients (43%) demonstrated a CR (1, 4%) or PR (11, 39%)(14).

Waldenstrom's Macroglobulinemia

Waldenstrom's Macroglobulinemia (WM) is a malignancy wherein Blymphocytes secrete excessive amounts of IgM antibodies. WM usuallyoccurs in people over sixty, but has been detected in adults in theirearly thirties. WM today is considered a rare incurable indolentmalignancy, which has in the past been treated by plasmaphoresis toreduce serum viscosity. Chemotherapeutic drugs such as an alkylatingagent and a corticosteroid are often prescribed. The most recommendeddrug for WM has been Leustatin (2CdA).

A report on seven patients with Waldenstrom's macroglobulinemia wherethe patients were treated with Rituximab® (375 mg/m² weekly times 4)(15)noted responses in 4 (57%) of patients. Median progression-free survivalwas 8 months (range 3-27+ months). Thus, Rituximab® should be useful incombined therapeutic protocols, particularly with chemotherapeuticreagents such as 2CdA.

Chronic Lymphocytic Leukemia (CLL)

CLL is the liquid (leukemic) equivalent of small lymphocytic lymphoma(SLL). Patients with SLL had lower serum levels and a lower responserate when treated with the standard dose of Rituximab® than patientswith other low-grade NHL subtypes. This is probably due to the very highlevels of circulating tumor cells in patients with CLL, and becausemalignant cells involved in CLL are thought to have reduced levels ofexpression of CD20 on the cell surface.

Nevertheless, the present inventors have discovered that hematologicmalignancies such as CLL may be treated with Rituximab®. A recentclinical study evaluated treatment of CLL patients at higher doses ofRituximab® (16). All patients receive a first dose of 375 mg/m³ tominimize infusion-relapsed side effects. Subsequent weekly dosages (3)remained the same but were given at an increased dose level. Sixteenpatients have been treated at dosages of 500-1500 mg/m³. Medium age was66 years (range, 25-78). Eighty-one percent had end-stage III-IVdisease. Medium white blood cell count was 40×10⁹/L (range, 4-200), Hgb11.6 g/dl (range, 7.7-14.7), platelets 75×10⁹/L (range, 16-160), medianβ₂ immunoglobulin was 4.5 mg/L (range, 3.1-9.2). Median numbers of priortherapies was 2.5 (range 1-9). Sixty percent of patients were refractoryto treatment. Two patients developed severe hypertension with the firstdose (375 mg/m³); another one received further therapy. Toxicity atsubsequent escalated dosages has been mild although no patient at the1500 mg/m³ dose level has been fully evaluated. Eight patients havecompleted therapy (4 at 500 mg/m³, 3 at 650 mg/m³, 1 at 825 mg/m³). Onepatient treated at 560 mg/m³ achieved full remission. One patient hasprogressive lymphocytosis on treatment and all other patients badreduction in peripheral blood lymphocytosis but less effect on lymphnodes. Dose escalation studies are ongoing.

Another approach to improving response in CLL patients is to upregulatethe CD20 antigen using cytokines. In an in vitro study, mononuclearcells from CLL patients were incubated for 24 hours with variouscytokines. Flow cytometry results showed significant up-regulation byIL-4, GM-CSF, and TNF-alpha (17). In fact, recent data suggests that theupregulation of CD20 observed on CLL cells may be limited to tumor cells(Venogopal et al. Poster—PanPacific Lymphoma meeting, June 1999.Cytokine-induced upregulation of CD20 antigen expression in chroniclymphocytic leukemia (CLL) cells may be limited to tumor cells).Preliminary data also suggest that interferon alpha also upregulatesCD20 on CLL cells after only 24 hours when applied at a concentration of500 to 1000 U/ml.

Thus, by administering certain cytokines to CLL patients prior to orconcurrently with administration of Rituximab®t, the expression of CD20on the surface of malignant B-cells may be upregulated, therebyrendering CD20, as well as other cell surface markers such as CD19, amore attractive target for immunotherapy. A collaborative study has beeninitiated to test for optimal cytokine doses for CD20 upregulation invivo. The study protocol involves treating ten patients initially withGM-CSF at 250 mcg/m² SQ QD X 3, ten patients with IL-4 mcg/kg SQ QD X 3,and ten patients with G-CSF at 5 mcg/kg SQ QD X 3. Mononuclear cellswill be separated by Ficon Hypaque centrifugation for apoptotic studiesto determine if upregulation of CD20 translates to enhanced killing oftumor cells by Rituximab®.

Antibody treatment of CLL can be combined with other conventionalchemotherapeutic treatments known to be useful for the treatment of CLL.The most frequently used single agent for CLL is chlorambucil(leukeran), given either as 0.1 mg/kg daily or 0.4 to 1.0 mg/kg every 4weeks. Chlorambucil is often combined with oral prednisone (30 to 100mg/m²¹d), which is useful in the management of autoimmune cytopenias.Cyclophosphamide is an alternative to chlorambucil, the usual dose being1-2 g/m² every 3-4 weeks together with vincristine and steroids (e.g.,COP regimen).

Various drug combinations have been used for CLL, including COP(cyclophosphamide, Oncovin, and prednisone), and CHOP (these three drugsplus doxorubicin). Fludarabine has shown an effect in the treatment ofCLL, and gave an ORR of 50% in a group of patients treated with 25-30mg/m²/d every 3-4 weeks. http://www.cancernetwork.com. Although somepatients have been shown to be refractory for fludarabine. Such patientsmay also be resistant to 2-CdA because often, patients who arerefractory to fludarabine are also refractory to 2-CDA (O'Brien et al.N. Engl. J. Med. 330: 319-322 (1994)).

Hence, anti-CD20 antibody therapy will be particularly useful forpatients who are refractory or who have relapsed after treatment withchemotherapeutic drugs. Rituximab® therapy may also be combined withradiotherapy in these patients. TBI with a low fraction size of 15 cGyto total doses of 75 to 150 cGy has been shown to be effective in aboutone-third of patients.

A Phase II trial is currently being conducted by CALGB in CLL patients.Rituximab® and fludarabine are administered concurrently, followed byRituximab® consolidation versus fludarabine induction followed byRituximab®. Rituximab® with Myeloablative Therapy Myeloablative therapyhas yielded responses in indolent lymphomas; however, residual tumorcells may remain despite high-dose therapy and the PBSC reinfused maycontain tumor cells. Rituximab® is being used before stem cellmobilization and after transplant to reduce residual CD20+ tumor cellsand contamination of the bone marrow or stem cells harvested. Interimresults demonstrated that no CD20+ cells were detectable in harvestedcells. Eighteen of 24 patients achieved engraftment and the treatmentwas well tolerated. PCR testing is ongoing to evaluate residual tumorcells (18).

Retreatment of Relapsed Low-Grade NHL with Rituximab®

A trial evaluating retreatment of 53 patients who had responded toRituximab® and later relapsed has been reported (19). Seven of 56evaluable patients (13%) obtained a CR and 16a PR (29%), for an ORR of42%. Four patients who had a second response received a third treatment;3 of these responded.

After treatment with two courses of Rituximab®, one patient's tumor,initially classified as follicular, small cleaved cell NHL, no longerexpressed the CD20 antigen and was unresponsive to Rituximab® at thetime of transformation to diffuse, large-cell NHL (20).

Thus, while retreatment with Rituximab® is effective for treatingpatients who have relapsed after prior treatment with Rituximab®, theremay be an increased incidence of CD20− tumor cells after secondarytreatment. This observation supports the utility of the combinedtherapeutic treatment regimens described herein.

Combination of Rituximab® and CHOP Chemotherapy for Low-Grade NHL

Chemotherapy with cyclophosphamide, doxorubicin, vincristine, andprednisone (CHOP) is an effective first-line therapy for low-grade orfollicular NHL. Though initial response rates are high, relapseeventually occurs and subsequent chemotherapy regimens produceremissions with shorter durations. A Phase II trial was initiated toevaluate the combination of CHOP and Rituximab® (21) in newly diagnosedand relapsed low-grade or follicular NHL because their mechanisms ofaction are not cross-resistant, and Rituximab® is synergistic withcertain cytotoxic drugs, including doxorubicin (5).

Twenty-nine of 38 patients received no prior anticancer therapy. CHOPwas administered at standard doses every three weeks for six cycles withsix infusions of Rituximab® (375 mg/m²). Rituximab® infusions 1 and 2were administered on Days 1 and 6 before the first CHOP cycle, whichstarted on Day 8. Rituximab® infusions 3 and 4 were given 2 days beforethe third and fifth CHOP cycles, respectively, and infusions 5 and 6were given on Days 134 and 141, respectively, after the sixth CHOPcycle.

In this combination study, 100% of the 38 patients treated responded(CR, 58%; PR, 42%). Of 35 evaluable patients who completed treatment,there were 63% CR, and 37% PR (21). Median DR is 35.3+ months withmedian progression-free survival not reached after a median observationtime of 36.7+ months. Twenty patients are still in remission after 36+months to 53.4+ months (22). This DR is impressive even for first-linetreatment, and 24% of this trial population had relapsed afterchemotherapy.

In a study to be conducted by CALGB, 40 patients with low-grade NHL willreceive Rituximab® weekly times 8 and oral cyclophosphamide dailystarting on Day 8. Twenty patients will receive Rituximab® alone for 8weekly doses.

A Phase III study conducted by ECOG in patients with low-grade NHL iscomparing the combination of cyclophosphamide and fludarabine (Arm A)with standard CVP therapy (Arm B). In the randomization to Arm A or ArmB, patients are stratified by age, tumor burden, histology, and Bsymptoms. Responders in both arms will undergo a second randomization toRituximab® maintenance therapy (375 mg/m² weekly times 4 every 6 monthsfor 2 years (Arm C) or to observation (Arm D).

Combination of Rituximab® with Cytokines

Rituximab® Plus Interferon Alpha

Interferon is a cytokine involved in modulating the immune system (23).Mechanisms by which interferon may increase the effectiveness ofantibodies include the potentiation of antigen expression (24),increased targeting of antibodies into tumors (25,26), and enhancedcytotoxicity of immunotoxins (27).

In a combination trial, interferon-alpha (Roferon-A), a cytokine with asingle-agent clinical activity in NHL (28), and Rituximab® were given topatients with relapsed low-grade or follicular NHL. Interferon-alpha(2.5 or 5 MIU) was administered subcutaneously, three times weekly for12 weeks. Rituximab® was administered by IV infusion weekly for fourdoses (375 mg/m²) starting on the fifth week of treatment. The ORR was45% ( 17/38 patients); 11% had a CR and 34% had a PR. Kaplan-Meierestimates of the median DR and TTP in responders were 22.3+ and 25.2+months, respectively (29). Previous combination studies ofinterferon-alpha and chemotherapeutic regimens containing anthracyclinesyielded prolonged time to progression, but did not consistently increaseresponse or survival rates (30-32). These early results suggest that thecombination of Rituximab® and interferon-alpha may prolong the time toprogression relative to Rituximab® alone.

Rituximab® Plus G-CSF

In a separate study, Rituximab® and G-CSF are being evaluated inrelapsed low-grade NHL. It has been demonstrated in vitro as well as invivo in healthy volunteers that G-CSF, via its effect on myeloidprecursor cells, induces FcRI-positive neutrophils that are capable offunctioning as effector cells in ADCC. Therefor, a Phase I/II study wasinitiated to evaluate the toxicity and efficacy of the combinedtreatment.

Both in Phase I and Phase II, patients were administered a standard doseof G-CSF (5 μg/kg/day) administered for three days, starting 2 daysbefore administration of Rituximab®. Phase I consisted of a doseescalation of Rituximab® (125, 250, or 375 mg/m² weekly X4). Earlyresults in 9 patients evaluated so far yielded an ORR of 67% (44% CR,22% PR) with minor toxicity in 8 of the 9 patients (33). The mostfrequent adverse events were fever ( 4/8 patients), rhinitis ( 4/8),chills (⅜) and headaches (⅜), which were comparable to the adverseevents observed previously in administration of Rituximab® alone. ThePhase II part of the study has been initiated, which will examine theefficacy of the combination of G-CSF and 375 mg/m² Rituximab® X4.

Rituximab® Plus IL-2

High-dose therapy with autologous peripheral blood stem cells (PBSC) orbone marrow (BM) rescue has been used to treat NHL, however successremains limited by the high risk of relapse, which is 50-80%. In aneffort to improve durable remissions post-transplant, immunotherapyincluding high dose and low dose therapy with IL-2 has been studied in anumber of treatment centers. Such studies have suggested that IL-2therapy does demonstrate early post-transplant anti-Tumor activity.

Initially following autologous transplant patients display delayedimmune reconstitution which potentially results in diminishedimmune-mediated tumor eradication (43, 44). Indeed, it has been shownthat both CD$+T cells and cytotoxic CD8+ T cells are depressed (45-49).In vitro assays have demonstrated a profound suppression of T cellcytolytic and proliferative responses as well as decreased production ofIL-2 in response to mitogens and soluble antigens. However, soluble IL-2is able to restore these immune responses suggesting that immune cellsin patients after autologous transplant are capable of responding toexogenous IL-2 (47). Peripheral blood NK activity also remains lowerfollowing BMT than control values and the NK activity is also augmentedby addition of exogenous IL-2 (49). These data suggest thatadministration of IL-2 to patients shortly after stem cell transplantmay enhance immune responsiveness at a critical period when tumor burdenis minimal and when immune responsiveness in the absence of IL-2 islacking.

For instance, Caligiuru et al. have shown that IL-2 (Hoffman-LaRoche)administered at 0.45×10⁶ U/M²/day by 24 hour CIV for 12 weeks was ableto expand the absolute number of CD56 bright NK cells (50-52). Thisregimen was administered to non-transplant patients in the outpatientsetting with little toxicity.

Animal models have shown that non-LAK inducing low doses of IL-2dramatically enhances anti-tumor activity when administered withtumor-specific T effector cells (53). In addition, Soiffer et al. (54)administered low doses of IL-2 to 13 autologous BMT or T cell depletedallogeneic BMT recipients undergoing treatment for relapsed leukemia orlymphoma. Enhanced immunological responsiveness was demonstrated in thelaboratory with a 5- to 40-fold increase in circulating CD56 brightCD16+ CD3− NK cells. Moreover, this low dose regimen of IL-2 resulted inaugmented in vitro killing of the NK targets K562. When Soiffer et al.(55) updated the outcome of 29 allogeneic BMT patients who received lowdose IL-2, they found superior survival for these patients (70%)compared to histological controls (30%, p=0.41).

Lauria et al. (56) treated 11 patients with high grade NHL at a medianof 42 days after ABMT with IL-2 at a dose of 2×10⁶ IU/m² good for twoweeks and then 3×10⁶ IU/m² twice a week for a year. Phenotypic analysisshowed a persistent and significant (p=0.001) increase in the proportionand absolute number of total lymphocytes and especially of both CD16 andCD56 NK cells after 6 months of therapy. None of the patients progressedwith a median follow-up of twenty-two months (range 10-42 months) afterstarting therapy. In addition, two patients with residual disease afterABMT, one in the liver and second in the lymph nodes, obtained acomplete response after 7 and 10 months of IL-2 therapy.

Vey et al. (57) treated 25 patients with refractory or relapsed HD (11patients) and NHL (14 patients) with low dose IL-2.48% of the patientshad resistant disease at transplant and 84% achieved CR after ABMT. IL-2was started at a mean of 54 days after transplant and consisted of afirst cycle of 5 days followed by 4 cycles of 2 days every other week.Patients received a mean of 160×10⁶ IU/m² of IL-2. After a five yearfollow-up, the probability of survival and DFS is 72% (HD 73% and NHL70%) and 45% (HD 36% and NHL 48%).

A group at the Fred Hutchinson Cancer Research Center (FHCRC) hasrecently found that low dose IL-2 therapy was well-tolerated in theoutpatient setting, and that remissions in patients treated with lowdose IL-2 tended to be longer than without IL-2 treatment. IL-2 therapywas associated with an increase in the number of certain populations ofimmune cells, including CD8+ CD69+ cells; CD16+ CD8+ cells; CD16+ CD69+cells; CD16+ CD56+ cells; CD 16+ CD 122+ cells; CD 16+Dr+ cells; andCD8+ CD56+ cells. There was also an increase in the expression of lyticactivity against the tumor targets K562 and Daudi, with a median of5.9-fold and 6.5-fold increase, respectively. Relapses, when theyoccurred, occurred at a median of 17.8 months after transplant, andtherefor remissions were reported to be characteristically longer thanwhat was historically seen in transplant recipients without IL-2therapy.

Given the encouraging data gathered from single therapy studies withIL-2 on ABMT transplant recipients, it seemed reasonable to combine IL-2therapy with Rituximab® post transplant, given that Rituximab'sbiological activity appears to be mediated through ADCC andcomplement-mediated lytic activity. Thus, a Phase I trial has beeninitiated in collaboration with the FHCRC to evaluate the safety andpotential efficacy of a combined therapeutic regimen.

A separate Phase II study is also being performed to evaluate theefficacy and the incidence of HACA formation in patients receivinglow-dose IL-2 and Rituxan®. A specific objective of this study is toassess whether ADCC is enhanced by in vivo exposure to IL-2 and whetherADCC activity correlates with clinical response. Inclusion criteria forpatients are histologically confirmed stage II-IV low-grade, follicularB-cell or mantle cell lymphoma. Mantle cell lymphoma, for the purposesof this clinical study, is defined as CD5+, CD23− (if available) and/orbcl-1+ by immunohistochemistry. Patients who did not respond to or haverelapsed following their first treatment with a standard therapy, i.e.,chemotherapy, radiotherapy, ABMT and/or immunotherapy, are eligible.

Rituximab® plus GM-CSF for the Treatment of Relapsed Low Grade orFollicular B-Cell Lymphoma

Two separate Phase II trials have also been initiated to test theefficacy of combined treatment with Rituximab® and GM-CSF. One studyinvolves 40 patients with relapsed low grade B-cell lymphoma, andcomprises administering Rituximab® at 375 mg/m² weekly X 4 (d. 1, 8, 15,22) and GM-CSF (Leukine, Immunex) at 250 mcg sc three times weekly for 8weeks, starting one hour before the first dose of Rituximab®. This studywill be used to evaluate the clinical efficacy (overall response rate(ORR), overall complete response rate, time to progression andfailure-free survival) of the combined therapeutic regimen, tocharacterize the safety (qualitative, quantitative, duration andreversibility of adverse events) of the combined therapy, and todetermine the effects of the combined therapy on relevant lymphocytesubsets and cytokines. The second study plans to also monitorimmunologic parameters to assess the mechanism of killing (complement C3and C4, CH50, flow cytometry for CD3, CD4, CD8, CD16, CD19 and CD56 andADCC assay).

Rituximab® Plus Gamma-Interferon

Gamma-interferon may also be useful in combined therapy with Rituximab®for treating patients with low-grade or higher-grade lymphomas. It ishas recently been found that gamma-interferon upregulates CD20expression on multiple myeloma (MM) patient plasma cells, patientB-cells, as well as on normal donor B-cells (Treon et al., Lugano,1999). In fact, Treon and colleagues have shown that gamma-interferonaugments binding of these cells to Rituximab®. Induction of CD20expression on plasma cells occurred in a dose dependent manner, withupregulation seen with as little as 1 U/ml of interferon gamma. Aplateau occurred at 100 U/ml at 48 hours. Thus, gamma-interferon mayalso be beneficial when administered in combination with Rituximab®.

Intermediate-Grade and High-Grade NHL

Single-Agent Studies

In a study conducted in Europe and Australia, alternative dosingschedules were evaluated in 54 relapsed or refractory intermediate- orhigh-grade NHL patients (34). Rituximab® was infused at 375 mg/m² weeklyfor 8 doses or at 375 mg/m² once followed by 500 mg/m² weekly for 7doses. The ORR was 31%; (CR 9%, PR 22%) no significant differencebetween the dosing regimens was observed. Patients with diffuselarge-cell lymphoma (N=30) had an ORR of 37% and those with mantle-celllymphoma (N=12) had an ORR of 33%.

Combination of Rituximab® and CHOP Chemotherapy

In another study, 31 patients with intermediate- or high-grade NHL (19females, 12 males, median age 49) received Rituximab® on Day 1 of eachof six 21-day cycles of CHOP (35). Of 30 evaluable patients, there were19 CR (63%) and 10 PR (330%), for an ORR of 96%. This regimen wasconsidered well tolerated and may result in higher response rates thanwith Rituximab® or CHOP alone.

The NCI Division of Cancer Treatment and Diagnosis is collaborating withIDEC Pharmaceuticals Corporation to explore Rituximab® treatment inother indications. A Phase II trial of CHOP versus CHOP and Rituximab®is being conducted by ECOG, CALGB, and SWOG in older patients (>60years) with mixed, diffuse large cell, and immunoblastic large cellhistology NHL (N=630 planned). This study includes a secondaryrandomization to maintenance with Rituximab® versus non-maintenance.

A Phase III trial of Rituximab® and CHOP in 40 patients with previouslyuntreated mantle-cell lymphoma is also ongoing at the Dana FarberInstitute. Rituximab® is administered on Day 1 and CHOP is given on Days1-3 every 21 days for 6 cycles. Accrual for this study has beencompleted. A Phase II trial of CHOP followed by Rituximab® in newlydiagnosed follicular lymphoma conducted by SWOG has also been completed.Results of these two trials are being analyzed.

A Phase II trial of CHOP and Rituximab® versus CHOP alone in HIV-relatedNHL conducted by the AIDS Malignancy Consortium is ongoing; 120 patientsare planned.

Rituximab® after Myeloablative Therapy Relapse

Rituximab® has shown promising early results in patients with relapsedintermediate-grade NHL after high-dose therapy with autologous PBSCsupport. Six of seven patients responded (1 CR and 5 PR) and one patienthad stable disease; therapy was well tolerated (36).

Safety Experience

Adverse events and clinical laboratory data from 315 patients in thefive single-agent U.S. studies were combined to provide a safety profileof Rituximab® in patients with low-grade or follicular NHL. The majorityof adverse events were infusion-related and occurred with decreasingfrequency after the first infusion. The most common infusion-relatedevents were fever (49%), chills (32%), nausea (18%), fatigue (16%),headache (14%), angioedema (13%), pruritus (10%), and occasionally,hypotension (10%) and bronchospasm (8%). During the treatment period (upto 30 days following the last dose), 10% of patients experienced Grade 3or 4 adverse events, which were primarily infusion-related orhematologic. Thrombocytopenia (<50,000 platelets/mm³) occurred in 1.3%of patients, neutropenia (<1000/mm³) occurred in 1.9%, and anemia (<8gm/dL) occurred in 1.0%. Although Rituximab® induced B-cell depletion in70%-80% of patients, abnormally decreased serum immunoglobulins wereobserved in a minority of patients and the incidence of infection didnot appear to be increased.

Hypotension requiring interruption of the Rituximab® infusion occurredin 10% of patients and was Grade 3 or 4 in 1%. Angioedema was reportedin 13% of patients and was considered serious in one patient.Bronchospasm occurred in 8% of patients; 2% were treated withbronchodilators. A single report of bronchiolitis obliterans was noted.Most patients experienced no further infusion-related toxicities by thesecond and subsequent infusions. The percentage of patients reportingadverse events upon retreatment was similar to that reported followingthe first course (14).

Four patients developed arrhythmias during Rituximab® infusion. One ofthe four discontinued treatment because of ventricular tachycardia andsupraventricular tachycardias. The other three patients experiencedtrigeminy (N=1) and irregular pulse (N=2) and did not requirediscontinuation of therapy. Angina was reported during infusion andmyocardial infarction occurred four days post-infusion in one subjectwith a prior history of myocardial infarction.

The overall incidence of adverse events and Grade 3 and 4 adverse eventswas higher in patients with bulky disease than in patients withnon-bulky disease. The incidence of dizziness, neutropenia,thrombocytopenia, myalgia, anemia, and chest pain was higher in patientswith lesions >10 cm. The incidence of Grade 3 or 4 neutropenia, anemia,hypotension, and dyspnea was also higher in patients with bulky diseasecompared with patients with lesions <10 cm (19).

Since FDA approval of Rituximab® for treatment of relapsed or refractorylow-grade or follicular NHL in 1997, an estimated 17,000 patients havebeen treated. In May, 1998, descriptions of eight post-marketing reportsof severe infusion-related adverse events associated with the use ofRituximab® that resulted in fatal outcomes were summarized. In seven ofthe eight fatalities, severe symptoms occurred during the firstRituximab® infusion. The cause of death was not reported or remainsunknown for two of the eight cases. Severe respiratory events, includinghypoxia, pulmonary infiltrates, or adult respiratory distress syndromecontributed to six of the eight reported deaths. One patient had apretreatment lymphocyte count of 600,000/mm³; another, a creatinine of8; a third, a respiratory rate of 40; and a fourth, pancytopenia.Patients with a high tumor burden or with a high number of circulatingmalignant cells may be at higher risk and these patients should bemonitored closely throughout each infusion.

Most of the adverse events recently described were previously observedin Rituximab® clinical studies. One notable exception is aninfusion-related syndrome associated with rapid tumor lysis, that wasreported in six patients with high numbers of circulating tumor cells(37,38). This syndrome was characterized by fever, rigors, bronchospasmwith associated hypoxemia, a rapid decline in peripheral lymphocytes,laboratory evidence of tumor destruction, and transient, severethrombocytopenia. These patients had diagnoses of B-prolymphocyticleukemia (N=2), chronic lymphocytic leukemia (N=2), mantle-cell lymphoma(N=1), or transformed NHL (N=1); all had elevated circulatinglymphocytes, bulky adenopathy, and organomegaly. Although five of thesesix patients required hospitalization, symptoms resolved and subsequentRituximab® treatments were well tolerated; the last patient refusedfurther therapy and died of progressive disease two weeks later.

In a separate report of seven patients with CLL and one patient withmantle-cell lymphoma, tumor lysis syndrome was observed after the firstRituximab® infusion in those patients with lymphocyte counts >10×10⁹ L(39).

Radioimmunotherapy with ⁹⁰Yttrium-Labeled Anti-CD20 Antibody inCombination with Rituximab®

Another therapeutic approach to NHL under evaluation is a radiolabeledanti-CD20 antibody (IDEC-Y2B8) in combination with Rituximab®. IDEC-Y2B8(⁹⁰Y-ibritumomab tiuxetan) is a murine IgG, kappa anti-CD20 antibodyconjugated to ⁹⁰Y via a chelator, MX-DTPA, which is covalently bound tothe antibody. Rituximab® (250 mg/m²) is administered prior to IDEC-Y2B8to deplete peripheral B lymphocytes and improve biodistribution of theradiolabeled antibody.

In a recently reported Phase I/II study (40-42), patients with low-gradeNHL (N 34), intermediate-grade NHL (N=14), or mantle-cell lymphoma (N=3)were treated with IDEC-Y2138. The median age was 60, 71% were male, and96% were Caucasian. Of 51 patients with relapsed or refractory NHL, 34(67%) responded to single doses of 0.2, 0.3, or 0.4 mCi/kg of IDEC-Y2B8.The ORR was 82% ( 28/34) for patients with low-grade or follicular NHLand was 43% ( 6/14) for patients with intermediate-grade lymphoma. Nopatients with mantle-cell disease responded.

A Phase III randomized study comparing IDEC-Y2B8 with Rituximab® (375mg/m² weekly times 4) for treatment of low-grade follicular ortransformed NHL patients is ongoing. Another Phase III trial is alsobeing conducted in patients with relapsed NHL who are refractory toRituximab®.

SUMMARY

In the absence of curative therapy for NHL, the objective of treatmentis to achieve control of the disease for a meaningful duration andprovide relief of tumor-related symptoms without undue toxicity.Treatment with Rituximab® is a brief, 22-day outpatient therapy withlimited adverse events in most patients. In clinical studies, 50% ofevaluable relapsed or chemotherapy refractory low-grade or follicularNHL patients achieved complete or partial responses. These responseswere durable without maintenance therapy; the median TTP for responderswas 13.2 months and the median DR was 11.6 months in the pivotal study.

Rituximab® is approved as a safe and effective treatment for patientswith relapsed low-grade or follicular B-cell NHL. It has significantclinical activity, a novel mechanism of action, and compares favorablywith alternative therapies in response rate and response duration.Completion of ongoing studies will verify the role of alternativeRituximab® regimens and Rituximab® in the treatment of other CD20+B-lymphocyte malignancies.

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1-31. (canceled)
 32. A method of treating rituximab-refractory B-cellnon-Hodgkin's lymphoma, comprising administering an anti-CD20 antibodyto a patient in an amount effective to treat the lymphoma.
 33. A methodaccording to claim 32, wherein the antibody comprises human constantregions.
 34. A method according to claim 32, wherein the antibody ishumanized.
 35. A method according to claim 32, further comprisingadministering chemotherapy to the patient.
 36. A method according toclaim 35, wherein the chemotherapy comprises therapy selected from thegroup consisting of CHOP, CVP, ICE, mitozantrone, cytarabine, DVP, ATRA,idarubicin, Hoelzer chemotherapy regime, La La chemotherapy regime,ABVD, CEOP, 2-CdA, FLAG & IDA, FLAG & IDA with subsequent G-CSFtreatment, VAD, M & P, C-Weekly, ABCM, MOPP, and DHAP.
 37. A methodaccording to claim 32, wherein the non-Hodgkin's lymphoma is low gradeor follicular lymphoma.
 38. A method of treating previously untreatedB-cell lymphoma in a human patient comprising administering anon-radiolabeled anti-CD20 antibody to the patient, wherein thenon-Hodgkin's lymphoma is bulky disease non-Hodgkin's lymphoma.
 39. Amethod according to claim 38, wherein the method further comprisesadministering chemotherapy to the patient.
 40. A method according toclaim 38, wherein the patient is resistant to one or morechemotherapeutic agents.
 41. A method of treating low grade B-cellnon-Hodgkin's lymphoma in a human patient comprising administering tothe patient CVP therapy followed by rituximab maintenance therapy,wherein the maintenance therapy comprises four weekly administrations ofrituximab at a dose of 375 mg/m² every 6 months.
 42. A method accordingto claim 42, wherein the patient exhibits a response to the CVP therapy.43. A method according to claim 42, wherein the maintenance therapy isprovided for 2 years.
 44. A method of treating low grade or follicularnon-Hodgkin's lymphoma comprising administering an anti-CD20 antibody toa human patient in combination with chemotherapy as a first-linetherapy, wherein the chemotherapy comprises administration of an agentselected from the group consisting of cyclophosphamide, doxorubicin,vincristine, and prednisone.
 45. A method according to claim 44, whereinthe chemotherapy comprises CHOP.
 46. A method according to claim 44,wherein the anti-CD20 antibody is rituximab.
 47. A method of extendingtime to progression by at least 13 months in patients with relapsed orrefractory, low grade or follicular, CD20-positive, B-cell non-Hodgkin'slymphoma, comprising administering four doses of 375 mg/m² of rituximabto the patients.
 48. A method according to claim 47, wherein thepatients exhibit an overall response rate to the administration of about48%.
 49. A method of treating low grade or follicular non-Hodgkin'slymphoma comprising administering rituximab in 8 weekly doses of 375mg/m² to a human patient.
 50. A method according to claim 49, furthercomprising administering cyclophosphamide to the patient.
 51. A methodof treating Waldenström's macroglobulinemia comprising administering ananti-CD20 antibody to a patient in an amount effective to treat theWaldenström's macroglobulinemia.
 52. A method according to claim 51,wherein the antibody therapy is combined with a chemotherapeuticprotocol.
 53. A method according to claim 52, wherein thechemotherapeutic protocol comprises administration of 2-CdA.
 54. Amethod according to claim 51, wherein the anti-CD20 antibody isrituximab.
 55. A method of treating mantle cell lymphoma comprisingadministering an amount of an anti-CD20 antibody effective to treat themantle cell lymphoma to a human patient.